Phosphoglucomutase: its role in the response of pancreatic islets to glucose epimers and anomers

Rat pancreatic islets display phosphoglucomutase activity. The velocity of glucose-1-phosphate conversion to glucose-6-phosphate is increased in a dose-related fashion by glucose-1,6-bisphosphate. The islet homogenate, like purified muscle phosphoglucomutase, also catalyzes the synthesis of glucose-1,6-bisphosphate from glucose-6-phosphate and fructose-1,6-bisphosphate. The rate of the latter reaction is about 10,000 times lower than that of glucose-1-phosphate conversion to glucose-6-phosphate in the presence of glucose-1,6-bisphosphate. D-glucose and D-mannose, but not D-galactose nor D-fructose, markedly increase the islet content in glucose-1,6-bisphosphate. Such a content is twice higher in islets exposed for 5 minutes to alpha-D-glucose than in islets exposed to beta-D-glucose. The process of glucose-1,6-bisphosphate synthesis, as catalyzed by the alpha-stereospecific phosphoglucomutase, may play a role in the metabolic and, hence, secretory responses of the islets to glucose epimers and anomers.     

Oscillatory synthesis of glucose 1,6-bisphosphate and frequency modulation of glycolytic oscillations in skeletal muscle extracts

Oscillatory behavior of glycolysis in cell-free extracts of rat skeletal muscle involves bursts of phosphofructokinase activity, due to autocatalytic activation by fructose-1,6-P2. Glucose-1,6-P2 similarly might activate phosphofructokinase in an autocatalytic manner, because it is produced in a side reaction of phosphofructokinase and in a side reaction of phosphoglucomutase using fructose-1,6-P2. When muscle extracts were provided with 1 mM ATP and 10 mM glucose, glucose-1,6-P2 accumulated in a stepwise, but monotonic, manner to 0.7 microM in 1 h. The stepwise increases occurred during the phases when fructose-1,6-P2 was available, consistent with glucose-1,6-P2 synthesis in the phosphoglucomutase side reaction. Addition of 5-20 microM glucose-1,6-P2 increased the frequency of the oscillations in a dose-dependent manner and progressively shortened the time interval before the first burst of phosphofructokinase activity. Addition of 30 microM glucose-1,6-P2 blocked the oscillations. The peak values of the [ATP]/[ADP] ratio were then eliminated, and the average [ATP]/[ADP] ratio was reduced by half. In the presence of higher, near physiological concentrations of ATP and citrate (which reduce the activation of phosphofructokinase by glucose-1,6-P2), high physiological concentrations of glucose-1,6-P2 (50-100 microM) increased the frequency of the oscillations and did not block them. We conclude that autocatalytic activation of phosphofructokinase by fructose-1,6-P2, but not by glucose-1,6-P2, is the mechanism generating the oscillations in muscle extracts. Glucose-1,6-P2 may nevertheless play a role in facilitating the initiation of the oscillations and in modulating their frequency.     

Potentiation by glucose metabolites of inositol trisphosphate-induced calcium mobilization in permeabilized rat pancreatic islets

Saponin-permeabilized rat pancreatic islets degraded exogenously added inositol 1,4,5-trisphosphate (IP3), and degradation was inhibited in the presence of either fructose 1,6-bisphosphate or diphosphoglycerate. The addition of either fructose-1,6-P2 or diphosphoglycerate to 45Ca2+-labeled permeabilized islets potentiated 45Ca2+ release caused by IP3 (by either exogenously added IP3 or IP3 generated endogenously in the presence of carbachol or guanosine 5'-3-O-(thio)triphosphate (GTP gamma S). The effect of diphosphoglycerate and fructose-1,6-P2 on 45Ca2+ release correlated well with the effects of these agents on the recovery of radioactivity in IP3. These results further support our previous proposal that in pancreatic islets intracellular calcium mobilization may be sustained in part via the inhibition of IP3 degradation by metabolites produced during stimulation with insulinotropic concentrations of glucose (Rana, R.S., Sekar, M.C., Hokin, L.E., and MacDonald, M.J. (1986) J. Biol. Chem. 261, 5237-5240).     

The enzymic synthesis of ribose-1,5-bisphosphate: studies of its role in metabolism

Ribose-1,5-bisphosphate is synthesized in a reaction that uses ribose-1(or 5)-P as the phosphoryl acceptor and the acyl-P of 3-phosphoglyceryl phosphate as the donor. Glucose-1,6-bisphosphate is synthesized in a similar reaction. The relative activity with the two substrates remains unchanged over almost 300-fold purification of the enzyme, indicating that glucose-1,6-bisphosphate synthase catalyzes both reactions. The relative V/Km values for alternative phosphoryl acceptors are ribose-1-P (1); glucose-1-P (0.30); mannose-1-P and ribose-5-P (0.11); glucose-6-P (0.10); 2-deoxyglucose-6-P (0.03); and 2-deoxyribose-5-P (0.02). Fructose-1- and 6-phosphates are not substrates. The synthesis of both ribose-1,5-bisphosphate and glucose-1,6-bisphosphate is inhibited by physiologically significant levels of fructose-1,6-bisphosphate, glycerate-2,3-bisphosphate, glycerate-3-phosphate, citrate, and inorganic phosphate. Ribose-1,5-bisphosphate is a strong activator of brain phosphofructokinase.     

Role of inosine 5'-phosphate in activating glucose-bisphosphatase

Glucose-bisphosphate (G1c-1,6-P2) phosphatase has been purified greater than 200-fold from the cytosol of mouse brain. As reported earlier, the enzyme requires inosine monophosphate (IMP) and Mg2+ for activity [Guha, S.K., & Rose, Z. B. (1982) J. Biol. Chem. 257, 6634-6637]. Kinetic parameters and the role of IMP have been further investigated. When Glc-1,6-P2 and IMP are both varied, double-reciprocal plots of the data form a parallel line pattern. With 2 mM Mg2+, the Km obtained for G1c-1,6-P2 is 20 microM and the Ka for IMP is 9 microM. Co2+, Mn2+, and Ni2+ activate less effectively than Mg2+. The apparent Ka for Mg2+ decreases with increasing G1c-1,6-P2, and the observed Km of G1c-1,6-P2 decreases with increasing Mg2+. The extrapolated value of the Ka of Mg2+ at infinite substrate is 86 microM. Mg2+ does not affect the Ka of IMP. The phosphatase activity is optimal at pH 7. The phosphatase is not completely specific since mannose 1,6-bisphosphate is hydrolyzed and guanosine monophosphate activates. However, fructose 1,6-bisphosphate is no more than a poor inhibitor, and adenine nucleotides are neither activators nor inhibitors. The products of the reaction are glucose-1-P and glucose-6-P, in a ratio of 2:3, and Pi. Both glucose-P's are competitive inhibitors with respect to IMP [Ki(glucose-1-P) = 5 microM; Ki(glucose-6-P) = 18 microM]. Neither glucose-P competes with G1c-1,6-P2. The demonstration of an exchange reaction between G1c-1,6-P2 and glucose-6-P is evidence for the phosphorylation of the enzyme by the substrate. The exchange reaction requires Mg2+ and is inhibited by IMP. The observation of the exchange reaction and its elimination by IMP indicates that the low level of phosphoglucomutase activity that remains with the phosphatase throughout purification is an inherent property of the phosphatase. The requirement of glucose-bisphosphatase for the nucleotide IMP is consistent with possible roles for both G1c-1,6-P2 and IMP in the control of the ATP level in the brain.     

Glucose-induced accumulation of glucose-1,6-bisphosphate in pancreatic islets : its possible role in the regulation of glycolysis

A rise in the extracellular concentration of glucose from zero to 5.6 and 16.7 mM caused a graded increase in the glucose-1,6-bisphosphate content of rat pancreatic islets. Glucose-1,6-bisphosphate activated phosphofructokinase in islet homogenates, when the reaction velocity was measured at low concentrations of fructose-6-phosphate. It is postulated that glucose-1, 6-bisphosphate participates, together with fructose-2,6-bisphosphate, in the regulation of glycolysis in intact islet cells.     

Influence of bradykinin on glucose 1,6-bisphosphate and cyclic GMP levels and on the activities of glucose 1,6-bisphosphatase, phosphofructokinase and phosphoglucomutase in muscle

The intracellular concentration of glucose-1,6-bisphosphate (Glc-1,6-P2) in rat tibialis anterior muscle was markedly decreased following the injection of bradykinin. Injection of bradykinin also induced a significant increase in the level of cyclic GMP in muscle. The activity of glucose-1,6-bisphosphatase, the enzyme that degrades Glc-1,6-P2, was markedly enhanced by bradykinin, which may account for the decrease in the level of Glc-1,6-P2. The decrease in Glc-1,6-P2, the potent activator of phosphofructokinase and phosphoglucomutase, was accompanied by a concomitant reduction in these enzymes' activities. The bradykinin-induced decrease in Glc-1,6-P2 and in the activity of phosphofructokinase, the rate-limiting enzyme in glycolysis, may be involved in the pathogenic influences of this hormone in various clinical conditions.     

Evidence for the presence of glucose cycling in pancreatic islets of the ob/ob mouse

Pancreatic islets from ob/ob mice incubated with 3H2O and 5.5 mM glucose formed 3H-labeled glucose, 74 picoatoms incorporated/islet/h. Sixty-three percent of the 3H was bound to carbon 2 of the glucose. The amount of glucose-6-P dephosphorylated to glucose, determined from this incorporation, was 48 pmol/islet/h. Glucose utilization, measured by the formation of 3H2O from [5-3H]glucose, was 72 pmol/islet/h. The amount of glucose dephosphorylated was then about 40% of that phosphorylated. Thus, glucose-6-P is dephosphorylated to glucose to a significant extent by intact islets in vitro and presumably by the beta cells of the islets. The extent of this glucose cycling, i.e. glucose----glucose-6-P----glucose, may play a role in determining the extent of glucose-induced insulin secretion.     

The synthesis of mannose 1-phosphate in brain

The interconversion of mannose-6-P and mannose-1-P in brain has been shown to be catalyzed by a distinct enzyme. The enzyme has been separated from most of the phosphoglucomutase activity of the brain. The residual phosphoglucomutase activity (less than 1%) may be associated with phosphomannomutase itself. Mannose-1,6-P2 or glucose-1,6-P2 is required for the reaction as well as a divalent cation (Mg2+ greater than Co2+ greater than Ni2+ greater than Mn2+). Glucose-1-P, glucose-6-P, and 2-deoxyglucose-6-P are also substrates or inhibitors. Other phosphorylated sugars tested, glucosamine-6-P, N-acetylglucosamine-6-P, galactose-6-P, fructose-6-P, ribose-5-P, and arabinose-5-P, do not affect the rate of the reaction when assayed in the presence of mannose-6-32P.     

Novel expression of liver FBPase in Langerhans islets of human and rat pancreas

Several reports have indicated the absence of gluconeogenic enzymes in pancreatic islet cells. In contrast, here we demonstrate that liver fructose-1,6-bisphosphatase (FBPase) is highly expressed both in human and rat pancreas. Interestingly, pancreatic FBPase is active and functional, and is inhibited by AMP and fructose-2,6-bisphosphate (Fru-2,6-P2). These results suggest that FBPase may participate as a component of a metabolic sensing mechanism present in the pancreas. Immunolocalization analysis showed that FBPase is expressed both in human and rat Langerhans islets, specifically in beta cells. In humans, FBPase was also located in the canaliculus and acinar cells. These results indicate that FBPase coupled with phosphofructokinase (PFK) plays a crucial role in the metabolism of pancreatic islet cells. The demonstration of gluconeogenic recycling of trioses as a new metabolic signaling pathway may contribute to our understanding of the differences between the insulin secretagogues trioses, fructose, and glucose in pancreas.     

Enzymatic characterization of the pancreatic islet-specific glucose-6-phosphatase-related protein (IGRP)

Glucose is the main physiological stimulus for insulin biosynthesis and secretion by pancreatic beta-cells. Glucose-6-phosphatase (G-6-Pase) catalyzes the dephosphorylation of glucose-6-phosphate to glucose, an opposite process to glucose utilization. G-6-Pase activity in pancreatic islets could therefore be an important factor in the control of glucose metabolism and, consequently, of glucose-dependent insulin secretion. While G-6-Pase activity has been shown to be present in pancreatic islets, the gene responsible for this activity has not been conclusively identified. A homolog of liver glucose-6-phosphatase (LG-6-Pase) specifically expressed in islets was described earlier; however, the authors could not demonstrate enzymatic activity for this protein. Here we present evidence that the previously identified islet-specific glucose-6-phosphatase-related protein (IGRP) is indeed the major islet glucose-6-phosphatase. IGRP overexpressed in insect cells possesses enzymatic activity comparable to the previously described G-6-Pase activity in islets. The K(m) and V(max) values determined using glucose-6-phosphate as the substrate were 0.45 mm and 32 nmol/mg/min by malachite green assay, and 0.29 mm and 77 nmol/mg/min by glucose oxidase/peroxidase coupling assay, respectively. High-throughput screening of a small molecule library led to the identification of an active compound that specifically inhibits IGRP enzymatic activity. Interestingly, this inhibitor did not affect LG-6-Pase activity, while conversely LG-6-Pase inhibitors did not affect IGRP activity. These data demonstrate that IGRP is likely the authentic islet-specific glucose-6-phosphatase catalytic subunit, and selective inhibitors to this molecule can be obtained. IGRP inhibitors may be an attractive new approach for the treatment of insulin secretion defects in type 2 diabetes.     

The participation of glucose-1,6-diphosphate in the regulation of hexokinase and phosphoglucomutase activities in brains of young and adult rats

1. The level of glucose-1,6-diphosphate (Glc-1,6-P2), the powerful regulator of carbohydrate metabolism, was found to be strikingly decreased in brains of adult rats (5 months of age) as compared to young (10-14 days of age). 2. This age-related decrease in Glc-1,6-P2, the potent inhibitor of hexokinase and activator of phosphoglucomutase, was accompanied by a correlated increase in the activity of hexokinase and a reduction in phosphoglucomutase. 3. Evidence is provided showing that Glc-1,6-P2 participates in the regulation of these enzymes' activities with age. 4. The age-related changes in Glc-1,6-P2 and in the enzymes' activities in brain were opposite to those which we previously found in skeletal muscle. 5. These results suggest that Glc-1,6-P2 is involved in the regulation of carbohydrate metabolism during growth in both brain and muscle, as well as in the interrelationship between these two tissues.     

A specific enzyme for glucose 1,6-bisphosphate synthesis.

The reaction: glycerate-1,3-P2 PLUS GLUCOSE-1-P YIELDS TO GLUCOSE-1,6-P2 plus glycerate-P is catalyzed by a distinct enzyme of mouse brain. A divalent metal requirement was shown when the enzyme was treated with imidazole and EDTA. Mg2+, Mn2+, Ca2+, Zn2+, Ni2+, Co2+, and Cd2+ were quite effective cofactors. The enzyme, in better than 50 percent yield, has been purified away from 99 percent of the phosphoglucomutase, phosphoglycrate mutase, and phosphofructokinase. Acetyl-P, ATP, enolpyruvate-P, creatine-P, and fructose-1,6-P2 are not phosphoryl donors. Glucose-6-P and mannose-1-P are good alternate acceptors. Mannose-6-P, galactose-Ps, and fructose-Ps have little or no acceptor activity. Strong inhibition was found with fructose-1,6-P2, glycerate-2,3-P2, enolpyruvate-P, and acetyl CoA. From the amount of activity and the kinetic constants of the purified enzyme it seems likely that this enzyme is responsible for the glucose-1,6-P2 synthesis of brain.     

Inhibition of mitochondrial and soluble hexokinase from various rat tissues by glucose 1,6-bisphosphate

Mitochondrial and soluble Type I and Type II hexokinase from various rat tissues differed in their susceptibility to inhibition by glucose-1,6-bisphosphate (Glc-1,6-P2). In tissues where Type I is the predominant form, the mitochondrial enzyme was less susceptible to inhibition by Glc-1,6-P2 than the soluble enzyme, especially at high Mg2+ concentration. In tissues where Type II is the predominant form, the mitochondrial enzyme was more susceptible to inhibition by Glc-1,6-P2 than the soluble enzyme, especially at low Mg2+ concentration. The results suggest that changes in the intracellular concentrations of Glc-1,6-P2 and Mg2+ under various conditions would affect the activity of the bound and soluble hexokinase from different tissues in a different manner.     

Effects of vanadate and insulin on glucose 1,6-P2 and fructose 2,6-P2 levels in rat skeletal muscle

Levels of glucose 1,6-P2 but not fructose 2,6-P2 were found decreased in skeletal muscle of alloxan-diabetic ketotic rats. Administration of both insulin and vanadate restored the altered values without affecting fructose 2,6-P2 concentrations. In normal rats, insulin increased muscle levels of both sugars, and vanadate decreased glucose 1,6-P2 without changing fructose 2,6-P2 levels. Enzymatic activities involved in glucose 1,6-P2 and fructose 2,6-P2 metabolism were not affected under any experimental condition.     

Biosynthesis of bacterial glycogen. The nature of the binding of substrates and effectors to ADP-glucose synthase.

Kinetic studies with ADP-glucose synthase show that 1,6-hexanediol bisphosphate (1,6-hexanediol-P2) is an effective activator that causes the enzyme to have a higher apparent affinity for ATP- and ADP-glucose than when fructose-1,6-P2 is the activator. Furthermore, in the presence of 1,6-hexanediol-P2, substrate saturation curves are hyperbolic shaped rather than sigmoidal shaped. CrATP behaves like a nonreactive analogue of ATP. Kinetic studies show that it is competitive with ATP. CrATP is not a competitive inhibitor of ADP-glucose. However, the combined addition of CrATP and glucose-1-P inhibits the enzyme competitively when ADP-glucose is the substrate. In binding experiments, CrATP, ATP, and fructose-P2 appear to bind to only half of the expected sites in the tetrameric enzyme, while ADP-glucose, the activators, pyridoxal-P and 1,6-hexanediol-P2, and the inhibitor, AMP, bind to four sites/tetrameric enzyme. Fructose-P2 inhibits 1,6-hexanediol-P2 binding, suggesting competition for the same sites. Glucose-1-P does not bind to the enzyme unless MgCl2 and CrATP are present and binds to four sites/tetrameric enzyme. Alternatively, CrATP in the presence of glucose-1-P binds to four sites/tetrameric enzyme. Thus, there are binding sites for the substrates, activators, and inhibitor located on each subunit and the binding sites can interact homotropically and heterotropically. ATP and fructose-P2 binding is synergistic showing heterotropic cooperativity. ATP and fructose-P2 must also be present together to effectively inhibit AMP binding. A mechanism is proposed which explains some of the kinetic and binding properties in terms of an asymmetry in the distribution of the conformational states of the four identical subunits.     

Methylglyoxal is an intermediate in the biosynthesis of 6-deoxy-5-ketofructose-1-phosphate: a precursor for aromatic amino acid biosynthesis in Methanocaldococcus jannaschii

A biosynthetic pathway is proposed for creating 6-deoxy-5-ketofructose-1-phosphate (DKFP), a precursor sugar for aromatic amino acid biosynthesis in Methanocaldococcus jannaschii. First, two possible routes were investigated to determine if a modified, established biosynthetic pathway could be responsible for generating 6-deoxyhexoses in M. jannaschii. Both the nucleoside diphosphate mannose pathway and a pathway involving nucleoside diphosphate derivatives of fructose-1-P, fructose-2-P, or fructose-1,6-bisP were tested and eliminated. The established pathways did not produce the expected intermediates nor did the anticipated enzymes have the predicted enzymatic activities. Because neither anticipated pathway could produce DKFP, M. jannaschii glucose-6-P metabolism was studied in detail to establish exactly how glucose-6-P is converted into DKFP. This detailed analysis showed that methylglyoxal and a fructose-1-P- or fructose-1,6-bisP-derived dihydroxyacetone-P fragment are key intermediates in DKFP production. Glucose-6-P readily converts to fructose-6-P, which in turn converts to fructose-1,6-bisP. Fructose-6-P and fructose-1,6-bisP convert into glyceraldehyde-3-P (Ga-P-3), which converts into methylglyoxal by a 2,3-elimination of phosphate. The MJ1585-derived enzyme catalyzes the condensation of methylglyoxal with a dihydroxyacetone-P fragment, which is derived from fructose-1-P and/or fructose-1,6-bisP, generating DKFP. The elimination of phosphate from Ga-P-3 proceeds by both enzymatic and chemical routes in cell extracts, producing sufficient concentrations of methylglyoxal to support the reaction. This work is the first report of methylglyoxal functioning in central metabolism.     

Euglycemic hyperinsulinemia increases glucose 1,6-bisphosphate in human skeletal muscle

1. The effects of physiologic concentrations of insulin on the contents of glucose 1,6-bisphosphate (glucose 1,6-P2) and regulators of glucose 1,6-P2 synthase in intact human skeletal muscle have been investigated. 2. Insulin increased glucose 1,6-P2 from a basal value of 70 +/- 6 to 135 +/- 12 mumol/kg dry wt (P less than 0.001). 3. Activation of synthase could not be associated with changes in its inhibitors (fructose 1,6-P2, Pi, citrate) or its substrate glucose 6-P.     

Purification and regulatory properties of pyruvate kinase from Veillonella parvula.

The nonglycolytic, anaerobic organism Veillonella parvula M4 has been shown to contain an active pyruvate kinase. The enzyme was purified 126-fold and was shown by disc-gel electrophoresis to contain only two faint contaminating bands. The purified enzyme had a pH optimum of 7.0 in the forward direction and exhibited sigmoidal kinetics at varying concentrations o-f phosphoenol pyruvate (PEP), adenosine 5'-monophosphate (AMP), and Mg-2+ ions with S0.5 values of 1.5, 2.0, and 2.4 mM, respectively. Substrate inhibition was observed above 4 m PEP. Hill plots gave slope values (n) of 4.4 (PEP), 2.8 (adenosine 5'-diphosphate), and 2.0 (Mg-2+), indicating a high degree of cooperativity. The enzyme was inhibited non-competitively by adenosine 5'-triphosphate (Ki = 3.4 mM), and this inhibition was only slightly affected by increasing concentration of Mg-2+ ions to 30 mM. Competitive inhibition was observed with 3-phosphoglycerate, malate, and 2,3-diphosphoglycerate but only at higher inhibitor concentrations. The enzyme was activated by glucose-6-phosphate (P), fructose-6-P, fructose-1,6-diphosphate (P2), dihydroxyacetone-P, and AMP; the Hill coefficients were 2.2, 1.8, 1.5, 2.1, and 2.0, respectively. The presence of each these metabolites caused substrate velocity curves to change from sigmoidal to hyperbolic curves, and each was accompanied by an increase in the maximum activity, e.g., AMP greater than fructose-1,6-P2 greater than dihydroxyacetone-P greater than glucose-6-P greater than fructose-6-P. The activation constants for fructose-1,6-P2, AMP, and glucose-6-P were 0.3, 1.1, and 5.3 mM, respectively. The effect of 5 mM fructose-1,6-P2 was significantly different from the other compounds in that this metabolite was inhibitory between 1.2 and 3 mM PEP. Above this concentration, fructose-1,6-P2 activated the enzyme and abolished substrate inhibition by PEP. The enzyme was not affected by glucose, glyceraldehyde-3-P, 2-phosphoglycerate, lactate, malate, fumerate, succinate, and cyclic AMP. The results suggest that the pyruvate kinase from V. parvula M4 plays a central role in the control of gluconeogenesis in this organism by regulating the concentration of PEP.